Abstract
This study aims to construct plasmid vectors useful to measure promoter strengths in cyanobacterium Synechococcus elongatus PCC 7942 and to analyze quantitatively various DNA sequences. Luciferase gene (luc) from firefly (Photinus pyralis) as a reporter was obtained as SD-containing luc gene from pSP-luc+NF (Promega) and cloned within a linker inserted between SacI and XhoI sites in pUC303, resulting in a promoter-probe vector, pLUC1. Various blunt-ended DNA fragments were cloned into SrfI site upstream the luc gene on pLUC1, which were used to transform E. coli MOSblue as well as streptomycin-resistant (rps12-R43) GRPS1 strain derived from S. elongatus PCC 7942 R2-SPc.
The psbAI promoters (193bp, 97bp, 55bp) from PCC 7942 and lacZ promoter (107bp) from E. coli were cloned into pLUC1, resulting in pLUC1-psbAI-193, -97, -55 and pLUC1-lacZ-107, respectively. Specific activities of luciferase in transformants of GRPS1 carrying recombinant plasmids allowed us to confine psbAI promoter regions that might interact with positive or negative regulatory factor(s).
